Cellulose constitutes the major storage form of photosynthesized glucose, and the major component of solar energy converted to biomass. World wide demand for energy and for food supplies are increasing. Cellulose is an attractive raw material for supplying these needs, because of its abundance. The glucose subunits of cellulose can be used in a variety of processes for production of energy on the one hand or for the production of protein on the other. A major difficulty which has stood athwart the advance of cellulose utilization technology has been the difficulty of obtaining glucose in reasonable yield from cellulose at a reasonable cost in terms of energy input, equipment requirements and the like. Enzyme-catalyzed hydrolysis of cellulose is an attractive potential solution to these difficulties. However, the production of adequate amounts of cellulase is dependent upon obtaining a suitable source of large quantities of the enzyme in a reasonably pure state.
Cellulases are found in the digestive tracts of snails, in certain anaerobic bacteria and in other microorganisms, for example the rumen microorganisms which inhabit digestive tracts of ruminants. A number of fungal species are known to produce cellulase, including fungi of the class Ascomycetes, such as Neurospora and Trichoderma. The fungal systems are perhaps the most attractive because the organisms can be cultured without resort to unusual growth conditions and some, at least, are capable of rapid growth.
The fungal system described herein is derived from Trichoderma reesei, herein T. reesei, an Ascomycete fungus species formerly assigned to the species Trichoderma viride. In general, any Ascomycete fungus capable of synthesizing a complete cellulase could be used to derive a strain having similar properties. T. reesei is presently preferred because large amounts of cellulase are produced extracellularly. See, Simmons, E. G., Abstracts of Second International Mycology Congress, Tampa, Fla., page 618 (1977). The cellulolytic system of enzymes by this species include an endo-.beta. glucanase, exo-.beta.-glucanase, and .beta.-glucosidase. The first of these enzymes is capable of hydrolyzing .beta.-glucosidic bonds at mainly internal sites on the cellulose molecule. The second is capable of catalyzing the hydrolytic removal of disaccharide subunits from the ends of the cellulose chain, yielding mainly cellobiose as a product. The .beta.-glucosidase catalyzes the hydrolysis of cellobiose to glucose. The term cellulase, as used herein, includes all such enzymes and their isozymes. The cellulase produced by T. reesei is found as soluble protein in the growth medium. Synthesis of cellulase by wild type T. reesei is under stringent metabolic and genetic control, in which both induction and repression are observed. The term induction is used herein to mean the presence of a substance necessary for the synthesis of the enzyme by the organism. Repression is a term used to describe the phenomenon in which the presence of a substrate in the growth medium is sufficient to prevent the synthesis of the enzyme. The presence of a repressor substance for a particular enzyme prevents the expression of the gene coding for that enzyme, and in some cases the presence of an inducer substance is additionally required for expression of the gene. In cultures of wild type T. reesei, cellulose acts as an inducer of the cellulolytic complex exclusive of .beta.-glucosidase and its presence is therefore required in the medium to obtain appreciable levels of these enzymes. A number of substrates act as repressors, notably glucose and glycerol. The necessary conditions for cellulase synthesis therefore are the presence of cellulose or other inducing substrates such as lactose and the near absence of glucose. However, as cellulase is synthesized and cellulose in the medium is degraded, glucose is produced, which may result in the repression of enzyme synthesis. Consequently, the levels of cellulase produced by the wild type strain are never very great. Furthermore, the synthesis of cellulase is characterized by a lag period due to the presence of a repressor substance. Once the growth medium has been exhausted of the repressor substance, synthesis of cellulase even in the presence of an inducer, does not begin for several hours. Consequently, maximal enzyme production requires mutational alteration of the wild type strain so as to lessen the stringent metabolic and genetic controls normally limiting the production of cellulase.